Chromosome replication, the single most important controlling event in the cell cycle, consists of initiation, polymerization, termination, and segregation steps. In Escherichia coli, polymerization is effected by DNA polymerase III holoenzyme and many other factors, including prepriming, priming, and single-strand binding proteins and helicases, topoisomerases, DNA polymerase I, and ligase. Among these polymerization factors is the dnaY gene product. This gene (or region) has been cloned, mutagenized, and sequenced. Surprisingly, a fragment of only 118 base pairs provides dnaY+ complementing activity (cloned in two different vectors and tested in dnaY(Ts) recA hosts). From sequence analysis and a study of in vivo transcription, it appears that the dnaY gene produces an RNA which could be the active product. It is proposed to determine the dnaY function by five subprojects. First, the dnaY RNA sequence will be determined and the secondary structure probed. Second, an in vitro complementation assay for dnaY will be developed. This will require the isolation of a group of new dnaY(Ts) mutants. The only available dnaY(Ts) mutant is leaky in vivo and extracts are not Ts in the M13 SS yield RF reaction (although M13 replication in vivo requires dnaY+ function). Third, the assay will be used to prove whether dnaY functions as a site, produces an active RNA product, or encodes a short polypeptide. Fourth, the specific biochemical step effected by the dnaY product will be determined. Fifth, the regulation of dnaY expression will be described. Chromosome replication is the key process in the cell cycle. When it is understood on a molecular level, it should be possible to understand and control the growth of normal and cancer cells. Because of the unexpected small size of the dnaY region and the possibilities that it could function as a site or produce an active RNA product, a study of dnaY might provide some important new clues about the replication mechanism.